Polioviruses are members of the Enterovirus genus of the family Picornaviridae. Polioviruses are small, non-enveloped viruses with capsids enclosing a single stranded, positive sense RNA genome. There are three types of polioviruses: types 1, 2, and 3. Infections of susceptible individuals by poliovirus can result in paralytic poliomyelitis. Poliomyelitis is highly contagious. Two different polio vaccines have been developed, the inactivated poliovirus vaccine (IPV) of Salk and the live, attenuated oral poliovirus vaccine (OPV) of Sabin. Both vaccines are safe and effective. Each has its particular advantages and disadvantages, and both have played an important role in the control of poliomyelitis. For a review about polioviruses and polio vaccines see, e.g., Kew et al., 2005.
Oral polio vaccine (OPV) is cheap and convenient, and has been used massively. However, occasional recipients suffer from vaccine-associated paralytic poliomyelitis (VAPP) due to revertants in the vaccine. Furthermore, it has been observed in populations that have not been fully immunized that the attenuated Sabin polio strains have undergone sufficient mutational changes to cause outbreaks of paralytic disease that are clinically and epidemiologically indistinguishable from naturally occurring wild-type polio disease; these mutants are called circulating vaccine-derived polioviruses or cVDPVs (see, e.g., Kew et al., 2005; Wright and Modlin, 2008; Yakovenko et al., 2009).
There is a growing consensus that inactivated poliovirus vaccine (IPV) may contribute to more rapid eradication of wild-type polio and control of emergent cVDPV when used in conjunction with existing OPV strategies (Wright and Modlin, 2008; John, 2009).
However, production of IPV is more expensive (see, e.g., John, 2009) and may even be prohibitively expensive for less and least developed countries, where a strong need for polio vaccines still exists. The culture systems for producing bulk poliovirus material that can be used in a vaccine, in particular non-attenuated poliovirus, contribute to a large extent to the relatively high costs.
Propagation of poliovirus in HEK293 cells has been described as a system for the study of neuron-specific replication phenotypes of poliovirus, and it was described that attenuated forms of poliovirus, such as poliovirus containing point mutations in an IRES element as present in the Sabin strains, demonstrated reduced propagation in HEK293 cells (Campbell et al., 2005).
E1-immortalized human embryonic retina (HER) cells, in particular PER.C6® cells, have been described as suitable for propagation of various viruses, with an emphasis on influenza virus (Pau et al., 2001; WO 01/38362, the contents of which are incorporated herein by this reference). Although WO 01/38362 describes working examples of propagation of various strains of influenza virus, and of Herpes Simplex Virus (HSV) types 1 and 2, measles virus and rotavirus in PER.C6® cells, propagation of poliovirus was not exemplified in WO 01/38362. Furthermore, the conditions for replication of poliovirus in such cells have not been described, and cannot easily be predicted based on replication of unrelated viruses in these cells. Hence, it was hitherto unknown whether it would be feasible to economically produce poliovirus at industrial scale for vaccine production purposes in these cells.
For large-scale manufacturing of inactivated polio vaccines, poliovirus is generally propagated in Vero cells, which are monkey-derived. Vero cells are widely used for vaccine production, including inactivated as well as live attenuated polio vaccines, and thus far are the most widely accepted continuous cell lines by regulatory authorities for the manufacture of viral vaccines, and use of these cells for vaccine production is expected to rise by experts in the field (Barrett et al., 2009).
Large-scale microcarrier culture of Vero cells for inactivated poliovirus vaccine has been described by Montagnon et al., 1982 and 1984. A process for the large-scale production of a polio vaccine using Vero cells, and the resulting vaccine, are also described in U.S. Pat. No. 4,525,349, the contents of which are incorporated herein by this reference.
High titers of poliovirus (Sabin type 1) production (almost 2×109 TCID50/ml) were described by (Merten et al., 1997) for conditions when Vero cells on microcarriers were cultured in serum-containing medium prior to the virus production phase in serum-free medium, but in view of the disadvantages of using serum these authors already indicate that a completely serum-free process is desired, and in such an optimized completely serum-free process these authors were able to obtain a titer of 6.3×108 TCID50/ml.
Kreeftenberg et al (2006), involved in production of poliovirus for vaccine production at industrial scale, also mention yields of various wild type and Sabin strains of poliovirus in Vero cells grown on micro-carriers, which yields are similar for the different strains, the log titers being between 8.1 and 8.6. These authors also describe that the amount of virus needed to produce the final vaccine is significantly higher for IPV than for OPV, which results in a significantly higher production cost per dose for IPV than for OPV.
Serum-free production of poliovirus using Vero cells cultivated on microcarriers has also been described by (Card et al., 2005), and although the level of productivity was lower than in static cultures, the microcarrier cultures were described as easier in scale-up.
Despite the efficacy and industrial applicability of these microcarrier-based Vero cell cultures, the production of large quantities of poliovirus remains costly.
Production of poliovirus using suspension Vero cells has been described, resulting in lower virus titers (10 log CCID50/ml between 6.5 and 7.9) than those observed in routine microcarrier Vero cells (van Eikenhorst et al., 2009).